The receptors are now being purified in sufficient quantities for amino acid sequencing of selected peptides. Those to be done this year include the N-termini of A and B and the DNA-binding domain. Specifically excluded from this effort are studies of the hormone-binding domain, for which we are seeking additional support in an independent effort, on which Dr. O'Malley is not a participant. Dr. Schrader will undertake that study independently. The native proteins are being recombined to study the stoichiometry of subunit assembly and to search for other potential factors regulating assembly. Key lysine amino groups are indicated, based upon blockade of assembly by reduction with NaBH4 of pyridoxal phosphate-receptor complexes. Photoaffinity labeled protein is being purified for determination of the amino acids constituting the active site for hormone binding. DNA binding studies now in progress include velocity sedimentation studies of natural gene-containing plasmids, effects of the receptors on hydrogen-bonding of DNA and conformational alterations in substrate accompanying binding. DNA sequence preference is sought by DNase protection and footprinting methods. Effects of this binding on gene transcription are tested for by changes in efficiency of RNA polymerase initiation in nuclei and DNA.